PROJECT SUMMARY TR&D Project 3. The Analysis Stage II: Tools for Analyzing the Connectivity and Morphology of Macromolecular Assemblies As the structure of an assembly is directly related to its functional role, we aim to define the architecture of isolated native macromolecular complexes of interest. For this, our molecular microscope pipeline needs information on the shape and connectivity of an assembly?s components, accurately representing their arrangement at the highest spatial and temporal resolution. Thus, we will develop and refine methodologies to determine the morphologies and spatial relationships between components within complexes at several distance scales, ranging from a description of the overall subunit shape and arrangement to defining atomic resolution contacts between pairs of macromolecules. Our strategy entails using orthologous methods, in order to provide complementary data and to cover a wide range of resolutions, to inform us about the shape, dimensions and connectivity of single proteins and macromolecular assemblies. We will focus on methods that have already proven particularly empowering, but which have significant scope for further advancement. These include electron microscopy (EM) and chemical cross-linking with mass spectrometry (XL-MS): through the former, we can produce morphological maps with sufficient detail to resolve the shapes and locations of complexes, proteins, domains and folds; in parallel, through the latter, we will obtain information on how each component of the assembly is positioned relative to all other components, and the entire structure. These data when combined with data from complementary, well-established methods, will be used to generate structural models of assemblies. To elucidate cellular functions, we propose to gather and interpret dynamic data about the changing morphologies of assemblies, and the changing interactions within these assemblies. Our molecular microscope pipeline thus seeks to build concrete high precision 3D models, and 4D models that change in time.